AU658890B2 - Method for detecting bovine diarrhoea virus infection, nucleotide sequence encoding a protein induced by this virus infection and recombinant proteins and antigens relating thereto - Google Patents

Abstract

In the method for detecting infection of a blood sample with a bovine viral diarrhoea (BVD) virus, there are detected, on the one hand, the anti-p80 antibodies by means of a recombinant antigen containing the BVD virus protein p80 produced in an eucaryotic host and, on the other hand, the presence of viral particles by means of antibodies directed against the BVD virus protein p80, in order to detect persistent viremias and acute infections with any BVD strain. Nucleotide sequence of BVD Osloss encoding the protein p80, recombinant proteins obtained from this sequence and recombinant antigens incorporating this recombinant protein. <IMAGE>

Description

658 9

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Rhone Merleux NB- ADDRESS FOR SERVICE: ytoactatte tutopeenne ce viot Innonegie DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Method for detecting bovine diarrhoea virus infection, nucleotide sequence encoding a protein induced by this virus infection and recombinant proteins and antigcns relating thereto The following statement is a full description of this invention, including the best method of performing it known to me/us:e oooeo o• 1° *ol -la- The present invention relates to a method for detecting bovine diarrhoea virus infection, a nucleotide sequence encoding a protein associated with this virus infection and recombinant proteins and antigens relating thereto.

Bovine diarrhoea virus (BVD) is an infectious single-stranded RNA-containing enveloped virus which is related to the conventional hog cholera virus and to the Border disease virus, the three viruses forming the Pestiviru. genus which belongs to the Togaviridae family.

The BVD virus is universally distributed in bovine populations and manifests itself by a wide range of clinical symptoms associated with congenital, respiratory or enter.' diseases (bovine viral diarrhoea, mucosal disease).

Isolates of BVD viruses may be classified into two distinct categories or biotypes according to their effects during cell culture: cytopathogenic and noncytopathogenic.

Acute infection of seronegative animals is normally benign or subclinical. On the other hand, intrauterine infection of the foetus, during approximately the first four months after the start of pregnancy, by a noncytopathogenic strain can not only produce abortions, still births or the birth of weak calves, but also the 30 birth of calves having persistent viremia, that is to say permanently excreting the virus. This period of four months corresponds to an absence of immunity in the foetus. When the immune system then becomes competent, it recognises the virus as its own and a situation of immunotolerance is established (absence of antibodies).

These animals will not be able to survive a subsequent infection by a cytopathogenic strain of homologous BVD virus.

2 Maintenance of the noncytopathogenic virus within the bovine population is ensured by its slow dissemination following acute infection of seronegative animals and, in particular, by its continual excretion by animals having persistent viremia. (See J. Brownlie et al., Ann.

Rech. Vet. (1987) 18:157-166).

A.Fenton et al. (Journal of Virological Methods, 27 (1990), 253-260) detect the Pestivirus antigens in the blood of viremic sheep infected in a persistent manner by the Border disease virus, by an ELISA carried out so as to detect a specific antigen in the leucocytes of these animals. This technique requires prior purification of the leucocytes, which proves to be long and complex to carry out.

The genome of the Osloss viral strain, of cytopathogenic biotype, has been cloned and completely sequenced by Renard et al. (Patent Application EP-A-0,208,672 of 8 July 1985). The Applicant has found that the open reading frame (ORF) of the BVD Osloss genomic sequence, which is 12408 nucleotides in length, has a coding capacity of 3951 amino acids (aas).

In an abstract distributed during the symposium on ruminant infections by Pestiviruses which was held at Hanover on 8 and 9 June 1990, C. Lecomte et al. indicate the identification of a cDNA translational product of the BVD virus immunoprecipitated by monoclonal antibodies •recognising the nonstructural protein p80 of a certain number of Pestivirus strains. The same cDNA is expressed in E. coli and the antigen produced is used in 30 competition ELISA to detect anti-BVD antibodies in the bovine serum.

The preparation and the characterisation of a series of monoclonal antibodies have been described by C. Lecomte et al. (Veterinary Microbiology, 23 (1990), 35 193-201), as well as the use of a fusion protein produced in E. coli as recombinant antigen enabling anti-BVD serum antibodies to be detected in a competition ELISA with the chosen monoclonal antibodies.

3 In an abstract distributed at the VIIIth International Congress of Virology which was held in Berlin on 26 to 31 August 1990, C. Lecomte et al. proposed the use of two ELISA tests for the detection on the one hand of anti-BVD antibodies and, on the other hand, of viral antigens. The anti-BVD antibodies in the serum would be detected by a competition ELISA using a BVD Osloss recombinant antigen p80 produced in E. coli and monoclonal antibodies specifically directed against the protein of a certain number of Pestivirus strains. The second ELISA would be a sandwich type ELISA using two monoclonal antibodies and which would permit the detection of antigens in persistent veremic animals.

However, the Applicant has found that the protein produced in E. coli is not recognised by all the monoclonal antibodies and especially by some of those exhibiting polyspecificity, that is to say reacting towards several or all Pestivirus strains, which undermines the chances of being able to detect in a single operation any infection by any BVD strain, all the more so as the second ELISA, based on the use of two monoclonal antibodies, could also not be sufficiently polyspecific.

The objective of the invention is to provide a very sensitive method of detection permitting a complete and effective control of infection which may be caused in livestock by a BVD virus of any type, and relating to both the detection of persistent viremias and acute infections.

30 The subject of the invention is therefore a method for detecting infection of a blood sample by the BVD virus, comprising a first test for the detection of anti-BVD antibodies and a second test for the detection "i *of viral particles, characterised in that the antibodies are detected by means of a recombinant antigen comprising the EVO virus nonstructural protein produced in a eukaryotic host, and preferably an monoclonal antibody used as competing antibody, and in 4 that the presence of viral particles is detected by means of polyclonal or monoclonal antibodies directed against the BVD virus protein p80, and preferably of a serum directed against the recombinant p80 antigen produced in a eukaryotic or prokaryotic host, for the detection of persistent viremias and acute infections by any BVD strain.

The p80 protein is preferably derived from BVD Osloss and the nucleotide sequence encoding this protein has been completely sequenced. The sequence is given in the list of attached sequences, under the reference SEQ ID No: 1. The Applicant has thus advantageously located two potential cleavage sites of p80 (KVR: lysine valine arginine) corresponding to the beginning and the end of The p80 protein is expressed in viral or eukaryotic vectors, and especially in the Baculovirus system, which is advantageously the baculovirus AcNPV (Autographa californica nuclear polyhedrosis virus).

The p80-encoding nucleotide sequence is introduced into an appropriate expression vector according to known techniques for constructing these vectors, especially those described in Patent Application EP-A-0,208,672..

Of course, the abovementioned nucleotide sequence includes all equivalent sequences, that is to say, which possess the essential properties of the sequence. By way of example, this would be the case for a sequence S".I encoding an identical amino acid sequence, but which 30 would use other specific codons by degeneration of the code. This would also be the case for a sequence encoding an amino acid sequence which is no longer identical but similar, taking into account the similarities between amino acids.

35 Equivalent sequence is also understood to mean a sequence derived from another BVD strain and maintaining recognition by anti-p80 antibodies.

t 5 The recombinant antigen is itself obtained from cultures of eukaryotic host cells which have been transfected with the p80-expressing vector, and preferably consists of extracts of these cells. The eukaryotic hosts may be animal or yeast, especially Saccharomyces cerevisiae, cell cultures. The transfer vectors for yeasts advantageously contain markers enabling useful recombinants to be selected for example by resistance to antibiotics or by other known means of selection (Broach J. et al., Meth. Enz. (1983) 101: 307). For the promoters, see also Hess et al., J. Adv. Enz. Reg. (1968) 7 149, and Holland et al., Biochemistry (1968) 17: 4900 or Itzeman et al., J. Biol. Chem..(1980) 255: 2073.

The animal cells are, preferably, known mammalian cell lines such as HeLa, CHO or BHX, insect cells, for example Spodoptera frugiperda (deposit ATCC CRL 1711, Sf9) (especially for the Baculovirus system) and, in general, lines whose use for the expression of substances to be administered to animals has been recognised by the health authorities, will be preferred. Viral promoters such as those of the SV40 virus (Fiers et al., Nature, (1978) 273:113) and of the CMV virus or human cytomegalovirus (McGregor and Caskey, Nucleic Acids Res.

17:2365, 1989),.or alternatively, that of the polyhedrin S 25 gene of the Baculovirus AcNPV or Autographa californica nuclear polyhedrosis virus (Hooft van Iddekinge et al., 1983, Virology 131: 561-565), will be used as promoter in these cellular constructs.

The recombinant antigen is preferably immobilised 30 on a solid support (for example microtitre plates), especially via an anti-p80 monoclonal antibody which is used as captor. Dilutions of bovine sera are placed in contact with the immobilised or nonimmobilised antigen and the anti-BVD antibodies are either directly revealed 35 by a bovine anti-IgG antiserum coupled for example to peroxidase or biotin (indirect ELISA), or revealed by competition ELISA with a second anti-p80 monoclonal antibody coupled for example to peroxidase or biotin. In 6 fact this detection may be carried out on any bovine blood fraction, especially serum and plasma.

Preferably, the viral particles in crude or white blood cell-enriched whole blood are revealed by mere centrifugation, especially for 30 minutes at 2500 g.

In order to ensure complete detection of the presence of viral particles of all the BVD types, a mixture of three p80-specific monoclonal antibodies is used as captor instead of a single monoclonal antibody.

A sandwich type ELISA is preferably carried out using, as captor, the mixture of three viral monoclonal antibodies, and as stain, the serum directed against the recombinant p80 protein. The serum is derived in particular from the immunisation of animals, in particular rabbits or goats, by repeated inoculations of recombinant p80 which may be produced both in prokaryotic and eukaryotic cells.

Samples corresponding to a viral titre of less than for example 10 3 pfu/ml (pfu plaque-forming units) may thus be detected as positive. In effect, the invention makes it possible to detect as positive, samples which, by the normal method of immunofluorescence on infected cells, may require three successive passages of the virus and the choice of appropriate host cells, which makes it necessary to propagate the virus on several cell types.

S" Since the p80 protein does not contain neutralisation epitopes, this method advantageously enables a '*distinction to be made between animals infected naturally 30 and animals vaccinated with a recombinant vaccine based on the structural proteins of the virus.

The subject of the invention is also the nucleotide sequence with the reference SEQ ID No: 1 which corresponds to the BVD Osloss sequence encoding the 35 nonstructural protein p80, or an equivalent sequence according to the definition given above, as well as any new nucleotide sequence containing it and comprising means permitting its expression or associated with 7 such means.

The subject of the invention is also the recombinant p80 protein corresponding to th- translation of this sequence, especially in a eukaryotic host, in the abovementioned expression systems, and any recombinant antigen containing this sequence, especially consisting of extracts of host cells, in particular eukaryotic cells, as stated above.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the position of the cDNA clones spanning the entire genome of the BVD virus (Osloss strain).

Figure 2 shows the analysis in vitro of the region of the BVD Osloss genome corresponding to the cDNA clone 174. The numbers therein indicate the size (base pairs bp) of the various fragments obtained by digestion of the 174 insert by each of the restriction enzymes used.

Figure 3 shows the location, on the BVD Osloss 20 genome, of the immunoreactive region of 80 amino acids and limits of the p80 protein.

Figure 4 shows the screening of epitopes in the immunoreactive region, which is 80 amino acids in length, situated within the p80 protein.

25 Figure 5 shows the map of the transfer vector pAcYMI described by Matsura et al. in J. Gen. Virol.

(1987), 68: 1233 to 1250.

I LOCATION AND SEQUENCING OF THE SEQUENCE ENCODING 30 1. The cDNA C174 was cloned (pcP 174 clone from the cDNA library described in European Patent Application -EP-A-0,208,672) (Figure 1) into the plasmid downstream of the RNA polymerase promoter of the bacteriophage SP6, between the EcoRI and BamHI sites (vector described by Melton D.A. et al., 1984, Nucleic Acids Res. 12:7035-7056).

2. After digestion with various restriction enzymes whose position of successive sites is indicated 8 in Figure 2, each fragment obtained was transcribed in vitro and then translated in an acellular system (lysate of rabbit reticulocytes). The translational products of increasing molecular weight were immunoprecipitated with anti-p80 monoclonal antibodies; this made it possible to locate an immunoreactive region amino acids in length, which is located in Figure 3 and whose sequence is given in the list of sequences under the reference SEQ ID No: 2.

3. The presence of recognised epitopes in this region of 80 amino acids was confirmed by epitope scanning, that is to say that 75 peptide hexamers spanning this region were synthesised.on a solid support and tested by ELISA in the presence of anti-p80 monoclonal antibodies and antibody-positive or negative bovine sera.

The result is illustrated in Figure 4.

4. Following the location of the epitopes, the sequence encoding the p80 protein was reconstituted so as to resemble the natural protein as 20 much as possible. The presence of two potential cleavage sites (KVR: lysine valine arginine) corresponding to the beginning and the end of p80 was determined by analysis of the genomic sequence of the BVD/Osloss virus and by comparison with that of known Flaviviridae; the S. 25 theoretical molecular weight of the protein between these two sites is 80430 daltons. The first of the two triplets occurs exactly at the same position in the genome of the BVD/NADL virus, and the entire BVD/Osloss genome contains only 5 KVR triplets all situated in the portion encoding 30 the nonstructural proteins of the virus (the NADL genome contains only three of them).

The p80 fragment of 2200 bp (base pairs) corresponding to the p80-encoding sequence was amplified by PCR (polymerase chain reaction) and cloned; its location is illustrated in Figure 3; its nucleotide sequence has the reference SEQ ID No:l.

9 II EXPRESSION OF THE RECOMBINANT ANTIGEN.

The integration of the p80 fragment into the genome of the Baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) was carried out via the transfer vector pAcYM1 described by Matsura et al.

Gen. Virol. 68: 1233-1250, 1987; restriction map in Figure 5) downstream of the promoter of the AcNPV polyhedrin gene. This vector contains the polyadenylation site of the polyhedrin gene as well as the gene for resistance to ampicillin and the replication origin of the plasmid pUC8 Messing, 1983, Meth. Enzymol.

101:20).

To this effect, the amplified fragment of the sequence SEQ ID No: 1 is digested with the restriction enzymes NcoI and BamHI and then treated with DNA polymerase (Klenow fragment) so as to make the 5' and 3'ends of the fragment blunt. It is then cloned into the BamHIdigested vector pAcYMl and treated with DNA polymerase (Klenow fragment) so as to make the ends blunt. The 20 recombinant plaimid pAcYM1-p80 is obtained, p80 being expressed under the control of the promoter of the polyhedrin gene.

The plasmid obtained is used to cotransfect the insect cells Sf9 together with the DNA purified from the 25 wild type AcNPV. A recombinant virus AcNPV-p80 was purified by plating the cotransfection supernatant and isolating the viral plaques by covering with agarose.

III ELISA TEST FOR DETECTING

ANTIBODIES

30 Principle: The test is based on the detection of anti-pb0 antibodies, in other words, antibodies directed against a nonstructural protein of 80,000 daltons associated with infection'by the BVD virus.

A recombinant antigen comprising the p80 protein derived from the Osloss virus is bound to a solid support viaa monoclonal antibody which is used as captor. The bovine blood or serum sample is placed ii, contact with 10 the bound antigen and is then either directly revealed by a coupled bovine anti-IgG antiserum, or placed in competition with a second coupled anti-p80 monoclonal antibody.

Materials, reagents and samples: 1. Materials and buffers: 96-well ELISA plates (NUNC MAXISORP), carbonate buffer, pH 9.6: 15 mM Na 2

CO

3 35 mM NaHCO3, 3 mM NaN3; .o was' buffers: PBS, PBS, 0.1% Tween -dilution buffer: PBS, 0.1% Tween 20, 10% horse serum (assessed to be free of BVD virus); saturation buffer: PBS, 10% horse serum (assessed to be free of BVD virus); chromogen and appropriate buffer.

2. Reagents: 1 anti-p80 monoclonal antibody used as captor of the antigen p80 (dilute ascites liquid); recombinant p80 antigen; 20 a) competition ELISA: 1 anti-p80 monoclonal antibody diluted and coupled to peroxidase o biotin; b) indirect ELISA: bovine anti-IgG serum (rabbit or goat) coupled to peroxidase or biotin.

3. Preparation of the samples: serum samples: collection of blood in tubes without anticoagulant; coagulation, minimum 4 hours at room temperature; centrifugation 2500 g, 30 minutes; collect the supernatant serum; 30 the test may be carried out on any blood fraction, especially plasma or even whole blood collected in tubes with anticoagulant.

Method: 1. Binding of the captor: The dilute anti-p80 monoclonal antibody is distributed in an amount of 100 pl per wall: contact overnight at 4°C; 3 washes with buffer 11 2. Saturation (optional): 150 pl/well of saturation buffer: 1 hour at 37"C.

3. Binding of the antigens recombinant p80 antigen (100 pl/well) contact overnight at 4 0

C.

4. Contact with the samples: 100 pl/well; sera diluted 10 x: contact 2 hours at 37"C, 3 washes with buffer Contact with the staining antibody: a) Competition ELISA: contact with dilute and coupled monoclonal antibody: 1 h 30 min at 37"C; 3 washes with buffer 3 washes with water.

b) Indirect ELISA: contact with dilute and coupled bovine anti-IgG serum; 1 h 30 r.in at 37 0

C;

20 3 washes with buffer 3 washes with water.

6. Staining with the chromogen: S..The procedure varies according to the chromogen and is detailed by its supplier.

IV ELISA TEST FOR DETECTING VIRAL PARTICLES.

Principle: The test is based on the detection of the protein, a nonstructural protein'of the BVD virus which is present in a large amount in the blood of infected animals. A mixture of three monoclonal antibodies specific for this p80 protein is used as captor and bound to the solid support (microtitre plates). Whole blood or the fraction of blood enriched with white blood cells or "buffy coat" is placed in contact with the bound captor mixture. A serum (rabbit or goat), directed against the recombinant p80 protein, is used as staining antibody either directly coupled (to peroxidase or biotin), or itself stained with a second antibody (rabbit or goat 12 anti-IgG) coupled to peroxidase or biotin.

The presence of viral antigen in the tested samples is indicated by the increase in the initial optical density in the presence of chromogen.

Materials, reagents and samples: 1. Materials and buffers: 96-well ELISA plates: NUNC-MAXISORP; carbonate buffer, pH 9.6: 15 mM Na 2

CO

3 35 mM NaHCO 3 3 mM NaN 3 wash buffer: PBS, 0.1% Tween saturation/dilution buffer: PBS, 0.1% Tween horse serum (assessed to be free of BVD virus); chromogen and appropriate buffer.

2. Reagents: mixture of 3 anti-p80 monoclonal antibodies, dilute ascites liquids; serum (rabbit or goat) directed against the recombinant p80 protein, either directly coupled (to peroxidase or biotin), or itself stained with a second antibody (rabbit or goat anti--IgG) coupled to peroxidase or biotin.

3. Preparation of the samples: Collection of blood in heparinised tubes; the 25 samples are tested either as they are, or after enrichment with white blood cells according to the following procedure: centrifugation at 2500 g; 30 minutes; after centrifugation, removing the upper layer consisting of plasma by pipetting, and collecting the buffy coat, a o 30 whitish zone situated between the plasma and the red blood cells.

Method: 1. Binding of the captor: Mixture of monoclonal antibodies diluted and distributed in an amount of 100 il per well; contact overnight at 4"C; 3 washes in PBS/Tween.

13 2. Saturation: 150 pl well of saturation buffer containing 10% of horse serum; 1 hour at 37"C.

3. Contact with the samples: 100 pl of whole blood or buffy coat per well; contact 2 hours at 37"C; 3 washes in PBS/Tween.

4. Contact with the staining antibody: Dilute anti-p80 sezum, coupled or uncoupled (peroxidase or biotin): contact 1 h at 37°C (100 pl/well); 3 washes in PBS/Tween; 3 washes with water; If uncoupled serum is used, repeat contact with coupled antiserum (1 hour at 37"C).

Staining with the chromogen: The procedure varies according to the chromogen chosen and is detailed by its supplier.

S.

*r 14 LIST OF SEQUENCES.

SEQ ID No: I.

TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 2236 base pairs NUMBER OF STRANDS: single CONFIGURATION: linear TYPE OF MOLECUL~E: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 40 2229 PROPERTIES: encodes the protein TOT AG AAT TTT GTT TAA OTT TAA GAA GGA CAT 4 .44~54 4 4 0 444444 4 4 4 4.

4* 4 4*4~ *4 *4 4 4 44* S 54 4 0*4 .4 4 4* 44 Gin

GAA

is

CAT

Th r

ACA

Tyr

TAT

Val1

GTA

Val.

QTN

Gly

GGA

Leu

CTA

Val

CIA

Gin

CAA

110

ATA

Th r

ACC

Val

GTG

Arg

AGA

Gly

GGT

Leu

TA

CAC

Th r

ACT

Pro

CCC

Gly

GA

Th r

ACC

Th r

ACC

GlY

GGA

Th r

ACC

Val

GIG

Val

GTC

Asn

MAT

Lou

CTC

Pro

CCA

11e

ATA

Ly s

MAG

Val.

GTC

Se r

AGO

I rp

TGG

Ala

GCA

Vial

TT

Ly s

AAG

Pro

CCA

Gin

CAA

Al a acO Phe

TTT

Asn

AAT

Se r

TCA

Met

ATG

Ala

C

Gl Y

GGC

Cy s

TGC

Th r

ACG

Gin

GAG

Ly s

AAA

lihe

TTC

Gi u

GAG

Giu

GAG

Lys~

AAA

Asri

AAC

TYr

TAO

LY S

A

Gin

CAA

Asp

GAO

Al a

GCA

Th r

ACA

Ph.

TT

Alat

CT

Gln

GMA

SerI

AGO

Arg

AGG

Thr

ACA

As p

GAO

Ser

ACT

Ser

TOO

Gly

GGT

As p

GAO

Se r

TCT

Se r

TOO

Th r

ACA

Gly

GGA

His

CAC

Len

CTA

Asn

AAC

01Y

GGA

Asn

AAT

Gly

GGG

Leu

CTG

So r

AGT

LY S

AAG

Al a

CC

Asop

GAO

Gin

CAA

Leu

CTG

As n

AAC

CYS

TGT

Ile

ATA

Glu

GAA

Lyb

AAA

Gly Pro ccc Asp

GAT

Phe

TTT

Gly

GOC

Val.

TT

Ls

AAG

Pro

CCA

Ser

ICA

Phe

TO

AkT A rg

AGG

Tb r

ACA

Lou

TTA

Lys

AAG

Gly

GC

Cy s TG7 Leu

TTA

Asp

GAT

Gly

GGG

Thr

ACA

Lau

TTG

Val

GTG

Ly s

AAA

Tb r

ACA

Gin

CAG

ATMA

Ile

ATA

ASP

GAT

Thi r

ACT

Gly

GGT

Ser

TOO

CYS

TOT

MAG

Val1

GCC

Len

TTA

G l.u

GAG

le AlA Ser Ser Val

AGO

Ser

ACT

As p

CAT

Al a

GCC

Ly s

AAG

Val

TT

Cl z

GGA

Cl y

GGC

Met

AIC

Met

ATG

Tb r

ACC

TCA

Met

ATG

Gin

GAG

A rg

AGG

Gly

OGA

Tb r

ACT

T r p

ICC

A rg

AGA

Ser

ACT

Val

GTC

Lau

OTT

CIA

Cly

GT

Thr

ACA

Cys

TGO

Ala

GOT

Ala

GCX

TCA

Val

GTT

Gly

GGT

Lys

MCG

Ala

GCA.

Asp

GAC

A rg

AG

Glu C AA Tyr

TAO

Ala

GOT

Ser

TOG

GGT

Ly s

XA

Ile

ATC

Ly s

AAG

Thr

ACA

Ile Met Gi' 1 Leu COO ATG QGA 010 N/CO 3 3 48 19 96 144 51 192 67 240 83 288 99 336 1 1 0 384 131 432 147 480 163 528 179 576 195 624 Gly Ala' Cly Lys Th:r Thr Glu Len Pro LYS GG GCA GGA AAA ACT ACA GAA CTC OCA AAG Ala Vial Ile Giu Glu OCA GTG ATA GAG GAG NTk 15 Gly

GGA

C;AG

AMn

AAC

Thi'

ACC

A rg

AGG

Cys

TGT

Ser

TCT

cGTA Glu

GAG

Glty

GG

GTA

Ly s

AAG

Leu Al a Gcc Th r

ACA

Phe 1'?C Al a

GCT

Arg

AGA

AGA

Ser

TCA

Le u

TTA

Tyr

TNG

Al a

CC

Ala

GCT

Glu,

GAA

Th r

ACT

Val

GTG

Leu

CT;\

Pro

CCC

Gly

GGG

A rg

AGG

Ilie

ATT

Gly

GGT

Ile

ATA

Gin

GAG

Se r AG C His

CAC

Val1

GTC

Arg

AGA

Al a

GICC

Ala

GCA

Thr

ACT

Ser

AGC

Th r

ACA

Met

ATC

Lys

AAA

Th r

ACP

Ty r

TAC

Val

GTG

Glu

GAG

Lau

CGG

Val G TA Ara

QGA

GInr

-CAG

Lys Arg A.AG CGG Tyr Gin TAT CAA Ile Gly ATA GGG Ser TYr TCA, TAT Met Val.

ATG tTA Pro Glu CCT GAG Ilie Ara ATA AGG Thrx, Gly ACA GGG Lys Gly AAA GGG Ile Pro ATC CCG Arc Asn AGA AAC Asn Ser AAC 2'CA Val Thr GTA ACA $or Gly TCA GGG L.ys Cys AAG TGT Thr Gly ACA GGC Ara Gly AGA GGC Giu Thr GAA ACA VlLou GTG CTA Tyr Met TAC ATG Asp Met G A C AIG Gly Tyr GGA TAT Giu Tyr GAG TAT Gin Leu CAG TTG Val. Val GTG GTT Gin Lys CAA AAA Glu Asp GAA GAC Val Giu GTT GAG Met Alat ATG GCA Gly Tyr OGG TAT Ser Gin TCN GAG Val Thr GTA ACG Glu Ly's GAA AAG Lou Lys OTT AAA Arg Val AGG GTA Ala Thr GCG ACC Val Lou Ile Pro L~eu 04

S..

S.

S S S S S S S *5*S *5 S S

S

S .5 G TG A ra

AGA

Ly5;

AAA

P he

TTT

Se r

TCA

Ala

GCT

Al a G CT His

CAC

Lau

CTT

GI U

GAG

Val1

GTT

Ty L,

TAC

Se r

TCO

Le u

CTG

A ra

AGG

A ra

AGA

Gly

GGT

Gly

GGG

Lou

TTG

clu

GAA

Cys

TGC

Tyr

TAC

Val

GTC

Met

ATG

Pro

CCA

Gly

GGA

Met

ATG

Asp

GAT

Ty r

TAC

Pro

CCA

Pro

CCA

Val1

GTG

Met

ATG

A rg

AGN

Se r

TCA

Lys5

AAA

Gly

GGG

Gin

CAA

Ile A TA 110

ATA

T hr

ACC

lie

ATA

Sear

AGC

Lys

AAG

Val

GTA

Set AC T Tyr

TAG

Asp

GAT

A ra AG G Ala

GCT

Val1

GTG

Lys

AAG

His

CAT

Asp

GAC

Met

ATG

Ph a

TTI

Gly

GGA

AlVL

GCC

Giu

GA

Gin

CAG

Gly

GGT

Ala

GCC

Gly

GGG

GTC

Le u

TTA

Val

GTG

Val1

GTC

MAG

ASP

GAC

Pro

CCC

Met

ATG

Pro CCa Lou

CTG

Lys

A

ACC

Glu

GMA

Phe

TTC

Asn

AAC

LYS

AAG

Glu

GAA

GTA

Asp

GAT

Se r

TCA

Th r

ACT

Pro cc Tyr

TAG

CTI ATA CCA TTG Arg

AGA

Set

AGT

Al c

GCA

Gin

CAG

As p

GAT

Ile AlT Pro C CA Ph he

TTC

Leu

GTT

Met

ATG

Lys

AAA

Asp

GAC

Val

GTA

Th r

ACA

Se r

TCA

Val

GTG

Gly

GGT

HIS

CAC

Ile

ATC

Thr

ACT

Pr-o

CCG

Glu

GAG

His

CAC

Ala

GCA

Ile As p

GAC

Lou

CGG

Leu

CTA

P ro

CCG

Al a G CC Val

GTT

Lys

AAA

Gly

GGC

Ara

AGG

Tyr

TAT

Ser

TOG

Gly G ("PG Lys

AAG

Tyr

TAT

Ara

AGA

Gly

GGG

Ala GCd Ile

ATA

Val

GTC

Lys

AAA

A~a

GCT

Th r

ACC

Val

GOTI

Ile

ATA

Giu G AA Tyr

TAC

Asp

GAG

P I*ic lTT Ile

ATC

Leu

GIG

His

CAC

P he lT So:'

TCA

Pro

CCT

Ala

GG

Phig I TC Al it

GCC

Asnr

AMG

Asn

AAT

Asp

GAC

Pro

CCT

Gin

GAG

Tyr

TAT

Lou

CG

Ala Ala Ala GCA GOA GCT 211 672 227 720 243 768 259 816 275 864 291 912 307 960 323 1008 339 1056 1104 371 1152 387 1200 403 1248 419 1296 435 3344 451 1392 467 1440 483 1488 499 1536 Lau Gln Ala TTA CAG GCA LeuGinAlaHis Arg Tyr Gly Ile Giu Asp Gly lie Asn Val Thr Lys TIAGAG CACGA AGG TAT GGG ATA GMA GAT GGA ATG AAG GIG ACA AAG 16 Ser Phe Arg Glu Met Xsn Tyr Asp Trp Ser [,eu Tyr Glu Glu Asp Ser 515 TCC TTT AGG GAA ATG AAT TAC GAT TGG AGC CTG TAO GAG GAG GAC AGO 1584 Lou Lou Ile Thr Gin Lou Glu Ile Leu As Asn Lou Lau Ile Sor Giu 531 TTG CTG ATA ACC CAG CTG GAG ATA CIG AAC AAT OTA CTC AIC TCT GAA 1632 Asp Leu Pro Ala Ala Val Lys Asn lie Met Ala Arg Thr Asp His Pro 547 GAC CIA CCA GCA GCA GTA AAA AAC ATC ATG GCA AGG ACT GAT CAC CCA 1660 Glu Pro Ile Gin Lou Ala Tyr Asn Ser Tyr Glu Val G)n Val Pro Val 563 GAA CCA ATC OAG OTT GCA tAC AAC AGT TAT GAG OTC CAG GTC CCT CIA 1728 Lau Phe Pro Lys-Ile Arg Asn Gly Glu Val Thr Asp Thr Tyr Glu Ash 579 CTG TT CCA AAA ATA AGG AAT GGG GAG GTT ACA GAT ACT TAC GAG AAC 1776 Tyr Sor Phe Leu Asn Ale Arg Lys Lou Gly Giu Asp Val Pro Val Tyry 595 TAC TCA TTC CIA AAT GCA AGA AAA CTA COG GAA GAT GTA CCT GTG TAO 1824 Ile Tyr Ala Thr Glu Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Lau 511 ATT TAT 0CC ACC GAA GAT GAA GAC CIG GCA GTA GAC QTT CTA GGC TTG 1872 Asp Trp Pro Asp Pro Gly Asn Gin Gin Val Val Giu Thr Gly Lys Ala 627 GAC TGG COC GAO CCA GGG AAO CAG CAA GTA GTG GAG ACT GGG AAA GCA 1920 Lau Lys Gin Val Val Gly Lou Ser Ser Ala Glu Asn Ala Leu Leu Ile 643 CTG MAG CAA GIG GIA GGA CTG TOC TOT OCT GAG AAT GCC CTG CTC ATA 1968 Ala Leu Phe Gly Tyr Val Gly Tyr Gin AlA Lou Ser Lys Arp His Val 659 GCC CTQ TTT GOG TAT GTA GGA TAT C A GCT TTG TCA AAM AGA CAC GTC 2016 0*00: Pro Met Ile Thr Asp lie Tyr Thr Ile Giu Asp Gin Arg Lou Giu Asp 675 CCA ATG AIC ACA GAC ATA TAC ACC ATA GAA GAT CAA AGA CTA GAG GAC 2064 0 Thr Thr His Lau Gin Tyr Ala Pro Asn Ala Ile Arg Thr Glu GXY Lys 691 ACA AOC CAC CTC CiA TAT GCA CCT MT GOT ATA AGA ACT GAG OGG AAG 2112 Glu Thr Glu Leu Lys G1U Leu Ala Val Gly Asp Met Asp Arg 1le Met 707 00 GAG ACT GMA CTA AAG GMA TIA GCA GTG GGT GAC ATG GAC AGA ATC ATC 2160 Olu Ser Ile Ser Asp TYr Ala Ser Gly Oly Lou Thr Phe Ile Arg Ser 723 GAA TCC ATC TCA GAT TAT GCA TCA GGA GGG TTG ACA TIC ATA AGA TOT 2208 r-rop Gin Ala Glu Lys Val Arg 2236 CAC 3CA GAG AM GTA CGC TAG COQ ATQ C

HI

17 SEQ ID No: 2 TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 240 base pairs NUMBER OF STRANDS: single CONFIGURATION: linear TYPE OF MOLECULE: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 1 240 PROPERTIES: encodes a p80 region of 80 amino acids comprising epitopes recognised by anti-p80 monoclonal antibodies.

GlY Tyr Gin Ala Leu Ser Lys Arg His Val Pio Met Ile Thr Asp Ile 16 GGA TAT CMA GOT TTG TCA AAA AGA CAC GTC CCA ATG ATC ACA GAC ATA 48 Tyr Thr Ile Glu Asp Gin Arg Leu Glu Aso Thr Thr Hi;s Leu Gin Tyr 32 TAO ACC ATA GAA GAT CMA AGA CTA GAG GAC ACA ACC CAC CIC CAA TAT 96 Ala Pro Asn Ala Ile Aro Thr Glu Gly Lys Glu Thr Glu Leu Lys GlU 48 GCA COT ANT GCT AlA AGA ACT GAG GGG MAG GAG ACT GAA CTA AAG GAA 144 Leu Ala Val Gly Asp Met Asp Aro Ile Mot Glu Ser 1l Ser Aso Tyr 64 TTA GCA GTG GGT GAC ATG GAG AGA ATC ATG GM TCC ATC TCA GAT TAT 192 Ala Ser Gly Gly Leu Thr Phe Ile Arp Ser Gin Ala Glu Lys Val Arg GCA TCA GGA GGG TTG ACA TTC ATA AGA TCT CAG GCA GAG AAA GTA AGA 240 lie *0 0 i~0

Claims (19)

1. Method for detecting infection of a blood sample by a bovine diarrhoea virus (BVD), comprising a first test for the detection of anti-BVD antibodies and a second test for the detection of viral particles, charac- terised in that the anti-p80 antibodies are detected by means of a recombinant antigen comprising the BVD virus protein p80 produced in a eukaryotic host, and in that the presence of viral particles is detected by means of Sub.sIfwi'atcUy 4Ae whole antibodies directed against -te BVD virus protein for the detection of persistent viremias and acute infections by any BVD strain.

2. Method according to Claim 1, characterised in that the recombinant p80 protein is encoded by the nucleotide sequence SEQ ID No: 1 or an equivalent sequence.

3. Method according to Claim 1 or 2, characterised in that the recombinant p80 protein is obtained from animal cell cultures transfected with a suitable vector 20 expressing the p80 protein.

4. Method according to Claim 3, characterised in that the recombinant p80 antigen is obtained from cultures of insect cells, such as Spodoptera frugiperda. Method according to Claim 2, characterised in that the recombinant p80 antigen is obtained from cul- tures of mammalian cells, such as HELA, CHO or BHK.

6. Method according to Claim 1 or 2, characterised in that the recombinant p80 antigen is obtained from cultures of yeast cells, such as Saccharomyces i 30 cerevisiae, transfected with a suitable vector expressing the p80 protein.

7. Method according to any one of Claims 1 to 6, characterised in that the recombinant p80 antigen com- prises extracts of eukaryotic host cells transfected with a suitable vector expressing the p80 protein.

8. Method according to any one of Claims 1 to 7, characterised in that the p80 protein is obtained by expression in a viral or eukaryotic vector. 19

9. Method according to Claim 8, characterised in that the p80 protein is expressed in the Baculovirus system, such as the Baculovirus AcNPV. Method according to any one of Claims 1 to 9, characterised in that the anti-p80 antibodies are detected in any bovine blood fraction, such as serum or plasma.

11. Method according to any one of Claims 1 to characterised in that the recombinant p80 antigen permit- ting the detection of antibodies, is immobilised on a solid support.

12. Method according to any one of Claims 1 to 11, characterised in that the presence of anti-p80 antibodies is revealed by a bovine anti-IgC antiserum coupled to a stain.

13. Method according to any one of Claims 1 to 11, characterised in that the presence of antibodies is revealed by a competition ELISA, in the presence of an anti-p80 monoclonal antibody coupled to a stain. 20 14. Method according to any one of Claims 1 to 13, "i characterised in that the presence of viral particles in crude or white blood cell-enriched whole blood is detected by mere centrifugation. Method according to any one of Claims 1 to 14, S 25 characterised in that the presence of viral particles is detected by means of a serum directed against the recom- binant p80 antigen produced in a eukaryotic or prokaryotic host.

16. Method according to any one of Claims 1 to 30 characterised in that, for the detection of viral particles, a mixture of three viral p80-specific mono- clonal antibodies is used.

17. Method according to Claims 15 and 16 as a whole, characterised in that a sandwich type ELISA is carried out with the mixture of monoclonal antibodies as captor and the serum as stain. 1.8. Nucleotide sequence with the reference SEQ ID No: 1 and corresponding to the BVD Osloss sequence encoding the nonstructural protein

19. Nucleotide sequence containing the sequence according to Claim 18. Recombinant protein corresponding to the translation of the nucleotide sequence according to Claim 18 or 19.

21. Recombinant protein according to Claim 20, characterised in that it is produced in a eukaryotic host transfected with a suitable vector expressing the said nucleotide sequence.

22. Recombinant protein according to Claim 21, characterised in that it is produced in Spodoptera frugiperda insect cells transfected with a B aculovirus vector such as the Baculovirus AcNPV.

23. Recombinant antigen containing the recombinant protein according to any •one of Claims 20 to 22.

24. Recombinant antigen according to Claim 23, characterised in that it 15 comprises etracts of host cells transfected with a vector expressing the said recombinant protein.

25. The method according to claim 1, the nucleotide sequence according to claim 18 or the recombinant protein according to claim 20 substantially as hereinbefore described with reference to any one of the Examples. Dated this 23rd day of January, 1995 S" *Rhone Merieux AND Societe Europeene de Biotechnologie by DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) 950123,p:\oper\jmw,18146-92.clm20 ABSTRACT A method for detecting infection of a blood sample by a bovine diarrhoea virus (BVD), comprises a first test for the detection of anti-BVD antibodies and a second test for the detection of viral particles, characterised in that the anti-pS0 antibodies are detected by means, of a recombinant antic[en comprising the BVD virus protein produced in a eukaryotic host, and in that the presence of viral particles is detercted by means of antibodies directed against the BVD virus protein p80. The method is useful for the detection of persistent viremias and acute infections by any BVD strain. OS FIG. 1